The morphology of animal genitalia and associated structures that function in sperm transfer is often very complex and divergent among closely related species, particularly in insects. For example, Drosophila melanogaster has 7 sibling species, among which the only reliable morphological characters for identification are the male genitalia. Because these secondary sexual characteristics appear to be among the first characters to change in the evolution of new species, they provide a number of advantages for investigating the genetic and molecular basis of interspecific differences in morphology. The proposed project has 3 specific aims. (1) One aim is to determine the genetic architecture of the differences in male genitalia between D. mauritiana and D. simulans, which belong to the melanogaster subgroup of sibling species. The approach is to mark approximately 70 segments of the genome of one species with P-element insertions containing eye color marker. Each segment will be introgressed individually into the genome of the other species by repeated backcrossing for 26 generations and then made homozygous. Analysis of variation in genitalic characters among these lines will provide an estimate of the number of genes that affect these traits and the relative magnitude of those effects. In some cases, the importance of dominance and epistasis will also be investigated. (2) Genes of D. melanogaster that are involved in the development of the male genitalia will be identified by enhancer detector screening and P- element mutagenesis. Enhancer detector insertions that show a different pattern of expression in male and female genital discs potentially identify genes involved in producing the sexually dimorphic genitalia. This potential will be investigated by inducing mutations in nearby genes through imprecise excisions of the P-element. (3) When loss of function genitalic mutants are found, their possible role in causing interspecific differences will be investigated through a hybridization test. Any gene that appears to contribute substantially to these differences will be cloned and its molecular structure characterized. The molecular basis of these differences can then be investigated by interspecific gene transfer using P-element transformation. With this information, one can begin to answer long standing questions about the relative importance of structural and regulatory genetic changes in morphological evolution.